Abstract A novel paradigm in paracrine signaling has recently emerged based on the findings identifying extracellular vesicles (EVs) as intercellular conveyors of biological information. Complementary to traditional modes of signaling, EV-mediated signaling appears to be critical for metabolic cooperation and coordination between cells, tissues and organs. In this context, extra-cellular RNA (exRNA), which includes microRNAs, circular RNAs, and long non-coding RNA or lncRNAs, contained both within in EVs as well as in complex with lipoproteins, have been shown by us and others to regulate gene expression and alter cell function in various cell types. Moreover, during pathological conditions such as cancer, the number and compositions of EVs and exRNA change, altering the host immune response as well as synchronizing the behavior of secondary tumors. Isolations and analysis of EVs and exRNAs both during normal and pathological conditions are critical for understanding EVs and exRNAs biogenesis and their effector functions. This information is a critical prerequisite for novel disease diagnostic and prognostic strategies, biomarker-based surveillance for disease progression, treatment efficacy and relapse. An important determinant in EVs biogenesis is represented by the complement system, which upon activation generates membrane-targeting complexes, MACs (membrane attack complex, C5b-9), which represent ungated Ca++ and water channels. Following complement activation, targeted cells either exocytose and endocytose of the MAC-containing areas of the plasma membrane generating in the process large number of EVs. Currently, the study of EVs as biological entities relevant for intercellular signaling and disease diagnosis is based on the assumption that the biogenesis of EVs and exRNA happen at a steady state rate, being modified mostly by the healthy/diseased status of the host. Our preliminary data strongly suggest that that may not be the case. Our results show that the tissue-origin, number, protein and RNA composition, of EVs isolated by standard techniques depend not just on the blood collection methods, but also on the time of day the blood samples were collected. In this application, we propose to establish a baseline for the quality, quantity and composition of extracellular RNA, taking into account the potential artifacts induced by the: 1) blood collection methods, 2) time of collection, 3) physical activity. Our results obtained with fresh samples, will be cross-validated using stored plasma samples from the Center for Clinical Research and Division of Transfusion Medicine (NIH Blood Bank).